Part:BBa_K1982012

VP64 transcription activitor

VP64
Function	transcription activator
RFC standard	RFC 10
Backbone	pSB1C3
Organism	synthetic construct
Source	synthetic construct
Submitted by	[http://2016.igem.org/Team:NEU-China NEU-China 2016]

Tetrameric VP16 transcription activator domain

Usage and Biology

VP64 is a transcriptional activator composed of four tandem copies of VP16 (Herpes Simplex Viral Protein 16). When fused to another protein domain that can bind near the promoter of a gene, VP64 acts as a strong transcriptional activator. This module is a classic molecular biology tool.

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)  CCGAAAAAGAAACGCAAAGTTGGGCGCGCCGACGCGCTGGACGATTTCGATCTCGACATGCTGGGT ... GACGACAAATAATAA  ORF from nucleotide position 58 to 240 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below) 1  MLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINYPYDVPDYASDYKDDDDK*
Sequence features: (with their position in the amino acid sequence, see the list of supported features) HA-tag:  44 to 52 Flag-tag:  54 to 61 Enterokinase cleavage site:  57 to 61
Amino acid composition: Ala (A) 4 (6.6%) Arg (R) 0 (0.0%) Asn (N) 1 (1.6%) Asp (D) 22 (36.1%) Cys (C) 0 (0.0%) Gln (Q) 0 (0.0%) Glu (E) 0 (0.0%) Gly (G) 3 (4.9%) His (H) 0 (0.0%) Ile (I) 1 (1.6%) Leu (L) 10 (16.4%) Lys (K) 2 (3.3%) Met (M) 4 (6.6%) Phe (F) 3 (4.9%) Pro (P) 2 (3.3%) Ser (S) 4 (6.6%) Thr (T) 0 (0.0%) Trp (W) 0 (0.0%) Tyr (Y) 4 (6.6%) Val (V) 1 (1.6%)
Amino acid counting Total number: 61 Positively charged (Arg+Lys): 2 (3.3%) Negatively charged (Asp+Glu): 22 (36.1%) Aromatic (Phe+His+Try+Tyr): 7 (11.5%)	Biochemical parameters Atomic composition: C298H434N64O115S4 Molecular mass [Da]: 6881.3 Theoretical pI: 3.19 Extinction coefficient at 280 nm [M-1 cm-1]: 5960 / 5960 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges
Codon usage Organism: E. coli B. subtilis S. cerevisiae A. thaliana P. patens Mammals Codon quality (CAI): good (0.69) good (0.74) good (0.64) good (0.69) excellent (0.86) good (0.77)
Alignments (obtained from PredictProtein.org)    There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.
Predictions (obtained from PredictProtein.org)
There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment.

Proof of function

Verification of the suppression efficiency of gRNA
With different target loci have been tested by the usage of a GFP reporter plasmid(pCold-1) with a CSPA promotor. The target sites can be determined by directing the gRNA consisting of 20 bp length against the desired sequence of interest. R1 with target sites at different distances to the promotor regions proved successfully as potential activation sites (see Table 1 and Figure 3).

Name	Binding Site	Distance to promoter	Sequence	Position
F1	CSPA promoter	15	TGCATCACCCGCCAATGCG	sense sequences
F2	non-coding	68	GCCGCCGCAAGGAATGGTG	sense sequences
R1	CSPA promoter	43	ATTAATCATAAATATGAAA	antisense sequences
R2	non-coding	94	CATCATCCAACTCCGGCAAC	antisense sequences

Table 1: Overview of the tested gRNAs with different binding sites on the GFP pCold-1 plasmid.

Figure 3: Position of the target loci on the GFP pCold-1 plasmid.

To ensure the suppression efficiency of the gRNA, four gRNA sequences targeting different sites of CSPA promoter were designed and transfected into the E. coli strains BL21. Efficient suppression of CSPA promoter in strains BL21 was observed. GFP levels in GFP transgenic strains decrease after inserting a fragment that expresses CSPA promoter gRNA. And the sequence with the best suppression effect was selected for further study.

Figure 4: Results of the GFP-influence under the CSPA promotor only using different gRNAs targeted to CSPA promoter in BL21.. 1-8: transfected with different GFP-gRNA plasmid (CSPA promoter) 9:Control group transfected with GFP plasmid,~27 kDa. Excitation wavelength: 488 nm, Emission wavelength: 509nm. Stationary cultures of BL21 was subcultured into fresh media and growth for 8 hours (1 3 5 7 9) or 16 hours (2 4 6 8) at 30°C. 30ng of protein with total volume of 30ul (protein sample + dissociation buffer).

Silencing capability validation

We next evaluated the effect of tCas9-cibn on suppressing CSPA promoter. GFP expression levels were assayed in strains BL21 after co-transformation with tCas9-cibn and CRY2-VP64 and gRNA in the absence of light conditions.

Figure 5: Silencing capability of tCas9-CIBN with gRNA using different gRNAs targeted to CSPA promoter in BL21. 1-8: transfected with different GFP-gRNA plasmid (CSPA promoter) and tCas9-CIBN plasmid(pBAD promoter) 9 10:Control groups transfected with GFP plasmid and tCas9-CIBN plasmid(pBAD promoter), ~27 kDa. Excitation wavelength: 488 nm, Emission wavelength: 509nm. Stationary cultures of BL21 was subcultured into fresh media and growth for 8 hours (1 3 5 7 9) or 16 hours (2 4 6 8 10) using 15mM L-arabinose at 30°C. 30ng of protein with total volume of 30ul (protein sample + dissociation buffer).

Compared with control groups, green fluorescence intensity and mRNA levels were dramatically reduced in groups treated with gRNA and tCas9-cibn. These results suggest that gRNA can specifically guide tCas9 to target upstream of CSPA promoter, thereby to inhibit CSPA promoter to reduce GFP expression levels.

Activation of a fluorescence reporter

Spatially controlled activation of gene expression was achieved in strains co-transfected with the LACE system, a reporter vector containing a gRNA target sequence upstream of CSPA promoter and the eGFP gene. Strains transfected with light-activated CRISPR/Cas9 effector (LACE) and incubated in the dark did not show a significant difference in eGFP levels compared to control groups transfected with empty plasmid. Strains containing the LACE system and gRNA exhibited significantly brighter eGFP fluorescence intensity when illuminated compared to when incubated in the dark.Activation of the eGFP reporter in strains transfected with the gRNA and LACE constructs, the gRNA and tCas9-VP64 expression plasmid or an empty plasmid as a negative control was quantified after 24 hours of illumination or incubation in the dark.

Figure 6: Activation capability of LACE system using gRNA-R1 plasmid and tCas9-CIBN plasmid in BL21. Excitation wavelength: 488 nm, Emission wavelength: 509nm. Stationary cultures of BL21 was subcultured into fresh media and growth for 8 hours using 15mM L-arabinose at 30°C.

Proof of expression

Stationary cultures of BL21 J23114 was subcultured into fresh media and growth for 4 hours. Subsequent Extraction of protein from Bacterial and visualization using SDS-PAGE confirms that proteins of the expected size are present in the supernatant and hence most likely successfully secreted by the engineered bacterial strains.

Figure 7: Western blot analysis of CRY2-VP64 protein levels. 1:Control groups transfected with empty plasmid; 2 3 4:transfected with CRY2-VP64 plasmid (J23114 promoter) from different single colonies, ~78.5 kDa. Stationary cultures of BL21 was subcultured into fresh media and growth for 4 hours at 30°C. 30ng of protein with total volume of 30ul (protein sample + dissociation buffer).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]